We are acquiring real time dynamics data from a sensitive laser scanning microscope system with a built-in long-term tissue incubation capability. A postdoctoral fellow Erik Martin has characterized RelA and c-Rel dynamics in two different cell lines with a panel of Toll-like receptor ligands using quantitative time lapse imaging (Martin EW et al. under revision). We used RNA-seq to identify gene clusters which are governed by specific dynamic features of RelA or c-Rel in the two cell types. We have also developed two novel knock-in mouse strains. Each strain expresses a fluorescent Rel (among the NF-B family of subunits) fusion from the native genomic locus, by CRISPR-Cas genome engineering. We screened pups for correct expression and integration of the targeting constructs. We are monitoring NF-kappaB dynamics alone or in parallel with cytokines or lineage TFs by crossbreeding the Rel knock-in mice with existing mice such as fluorescent reporters of cytokines or transcription factors associated with distinct phenotypes. Many of these factors are co-activated during immune cell differentiation. Moreover, simultaneous live cell imaging of two live reporters will reveal unprecedented insight about how separate dynamic networks interact with each other to achieve co-regulation of gene expression programs in immunity and cell differentiation. In another study (Martin EW et al. revised and submitted), postdoctoral fellows Erik Martin and Sayantan Chakraborty acquired high resolution quantitative live cell imaging data, indicating that RelA homodimers are more abundant forms of NF-kappaB proteins than previously thought.